Dopamine (DA) is involved in behaviors important for species survival including motivation, motor planning, sensorimotor integration, habit formation, reward prediction/valuation and neuroendocrine regulation, as well as in Parkinson's disease, schizophrenia, ADHD and drug addiction. Plasmalemma DA transporters (DATs) of dopaminergic neurons are crucial for terminating DA neurotransmission by removing extracellular DA. Because DAT' functions near synaptic areas of the neuronal plasma membrane, regulation of subcellular targeting and surface levels of DAT is critical for overall activity of DA systems. However, mechanisms controlling neuronal DAT localization and its trafficking/endocytosis are not well understood. In the previous cycle of this project, we defined mechanisms of protein kinase C-dependent endocytosis of DAT, discovered ubiquitination of DAT and revealed the role of the DAT amino-terminus in regulating its constitutive endocytosis. Many of these advances were made possible because we generated a functional epitope-tagged DAT with the hemagglutinin (HA) epitope inserted in the extracellular loop 2 of DAT. Overall, however, a significant limitation has been that most of our mechanistic understanding about DAT endocytosis is based on studies in model expression systems using non-neuronal cells. To study DAT endocytosis in DA neurons, we recently developed a transgenic knock-in mouse in which endogenous DAT was replaced by an HA-DAT. The HA-DAT mice have allowed us to begin quantitative analysis of endocytosis of the natively-expressed DAT and large-scale analysis of DAT interacting proteins. Development of this novel mouse model and new microscopy methodologies in our laboratory, combined with rapid advances in understanding the transporter structure, make it feasible - for the first time - to comprehensively characterize the dynamics of DAT movement and traffic in neurons and to determine how these dynamics are regulated by psychostimulant drugs of abuse such as cocaine, methylphenidate (MPH) and amphetamine (AMPH). The specific aims of the proposal are to: 1) define mechanisms that determine the subcellular localization and constitutive endocytic trafficking of DAT in neurons and the effects of the competitive DAT inhibitors cocaine and MPH on these processes; 2) define the mechanisms of DAT retention at the cell surface; and 3) elucidate the mechanisms by which signaling processes regulate neuronal DAT endocytosis. The proposed research will use a combination of the state-of- the-art quantitative cellular imaging, including electron microscopy, and mass-spectrometry.